Aims: Our overall objective is to understand the regulation of pro-inflammatory responses that develop in a spatially distributed, segment specific manner in the intact lung capillaries. Here, we will determine the role of mitochondrial mechanisms in regulation of leukocyte margination in arteriolar, septal and venular capillaries. The specific aims are to quantify for the first time capillary segment specific, regulation of endothelial (EC) mitochondrial (Ca2+mit) and cytosolic Ca2+cyt) (Specific Aim 1), generation of EC mitochondrial reactive oxygen species (ROS) (Specific Aim 2), and mitochondria-mediated leukocyte margination (Specific Aim 3). The new hypotheses to be tested are that mitochondrial distribution and the induction of mitochondrial mechanisms are vascular segment specific, and that differences in the extent to which mitochondrial mechanisms are invoked determine differences in pro-inflammatory responses in the various vascular segment of the lung. Procedures: 1) Morphometric measurements. Intravital imaging of mitochondrial and endosomal stores (ER) will be conducted in capillaries of the isolated, blood-perfused rat lung, using both conventional and confocal microscopy. 2) Ca2+ quantification, Ca2+mit, Ca2+cyt, and ER Ca2+ changes will be determined using flurophores that target the appropriate compartment. 3) ROS quantification. EC ROS production will be determined using fluorometric imaging of the ROS indicator dichloro fluorescin using our reported protocols. In one set of experiments we will test the hypotheses in a mouse lacking components of NADPH-oxidase to determine the role on non-mitochondiral ROS production. 4) Immunofluorescence imaging. Expression of P-selectin in capillaries will be determined using indirect in situ immunofluorescence imaging. 5) Leukocyte margination. Leukocyte margination will be determined using confocal and conventional microscopy using leukocytes labeled with, rhodamine 6G. 6) Ca2+ increase. EC Ca2+ will be increased by a) infusion of agonists and b) in situ photo-excited intracellular uncaging. Responses will be determined in terms of mitochondrial mechanisms that increase ROS production and leukocyte margination. Significance: This proposal addresses a new understanding of pro-inflammatory responses in different capillary segments. These segment-specific responses, which determine the preferential pathway for leukocyte margination during inflammation, are not adequately understood. It is important to understand the role of mitochondria, as mitochondrial mechanisms and mitochondrial ROS may critically regulate leukocyte margination and hence, lung injury. Mitochondrial ROS may also be involved in signaling gene transcription and consequently, lung remodeling. If the preliminary data hold, then this research will prove for the first time that mitochondrial mechanisms regulate vascular segment specific pro-inflammatory responses.